Structure of a eukaryotic cholinephosphotransferase-1 reveals mechanisms of substrate recognition and catalysis

Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cell membranes. In eukaryotes, two highly homologous enzymes, cholinephosphotransferase-1 (CHPT1) and choline/ethanolamine phosphotransferase-1 (CEPT1) catalyze the final step of de novo PC synthesis. CHPT1/CEPT1 joins two substrates, cytidine diphosphate-choline (CDP-choline) and diacylglycerol (DAG), to produce PC, and Mg2+ is required for the reaction. However, mechanisms of substrate recognition and catalysis remain unresolved. Here we report structures of a CHPT1 from Xenopus laevis (xlCHPT1) determined by cryo-electron microscopy to an overall resolution of ~3.2 Å. xlCHPT1 forms a homodimer, and each protomer has 10 transmembrane helices (TMs). The first 6 TMs carve out a cone-shaped enclosure in the membrane in which the catalysis occurs. The enclosure opens to the cytosolic side, where a CDP-choline and two Mg2+ are coordinated. The structures identify a catalytic site unique to eukaryotic CHPT1/CEPT1 and suggest an entryway for DAG. The structures also reveal an internal pseudo two-fold symmetry between TM3-6 and TM7-10, and suggest that CHPT1/CEPT1 may have evolved from their distant prokaryotic ancestors through gene duplication.


I would suggest a paragraph/figure on how the cryoEM determined structure compares to that predicted by AlphaFold2 2. On page 3 (Function of CHPT1) the first few sentences make it unclear if the CHPT1 from
Xenopus is 68% identical and 83% similar to both CHPT1 and CEPT1. As these are different genes I would separate and discuss percent similarity to human CHPT1 and human CEPT1 in separate sentences to clarify. 3. Methods (Cloning, expression and purification of Xenopus laevis CHPT1) -"condon" should be "codon" 4. Methods (Cloning, expression and purification of yeast pyrimidine…) -again, "condon" should be "codon" 5. Methods (Model building and refinement) -I believe "ploy-alanine" should be "polyalanine" 6. I might suggest a title with more gravitas, perhaps along the lines of "Determining how phosphatidylcholine is synthesized by structure and mechanistic studies of a eukaryotic cholinephosphotransferase"

Reviewer #2 (Remarks to the Author):
In this manuscript, the authors solve the first structure of a eukaryotic CHPT1, which carries out the final step of the Kennedy pathway of phosphatidylcholine biosynthesis. This is clearly an important target and is also a member of the CDP-alcohol superfamily of phosphatidyltransferases. The eukaryotic members have a different architecture than their prokaryotic counterparts and thus this work fills up an important void.

Key findings
The authors present two cryoEM structures of Xenopus laevis CHPT1, with CDP and CDPcholine bound in the active sites along with two Mg+2 ions. Although structures of related prokaryotic enzymes had been solved, the new structures reveal a novel architecture and enable the authors to propose a structure based mechanism of catalysis.
The authors have then done extensive mutational analysis of proposed functionally important residues demonstrating the expected effect on enzymatic activity. From their structures, the authors have also identified two residues, H133 and E129, hitherto not proposed as part of the signature motif. From docking, biochemical and sequence conservation, they propose these two be included in the signature motif for eukaryotic members of this superfamily.
The manuscript is written clearly and all the experiments have been planned carefully. The observations and conclusions are in agreement with the experimental data.
This manuscript in its current form requires a few points to be addressed before it can be considered for publication in Nature Comm.

One aspect that the authors dwell on at length is the dimerization. The dimerization surface seems to be fairly small and it is not clear if dimerization is -1) needed for biochemical activity and 2) is an artifact of surface delipidation during detergent extraction and purification. Although LMNG is not known to be a harsh detergent, such adventitious dimerization has been reported in the literature (see PNAS 103, 1723-1726 (2006)). So the following experiments would be important
Does the protein purify as a dimer in the presence of phospholipids (0.1 mg/ml.) added to the detergent in the buffer at the metal affinity and size-exclusion step ?
Can the dimer be broken by mutation of Y340 and K284 and thus disrupting the contacts ?
In the above cases, if the enzyme does indeed form a monomer, is it functionally active ? 2. Now that there are AlphaFold predicted structures of this enzymes, how does it compare with the experimental structure ? This is not to discredit the authors' structures, that contain substrates that are not in the AlphaFold prediction. However, this will be an important piece of information and should be included in the supplementary figures.
3. Although the authors discover and establish the importance of H233 and E129, they haven't proposed a unifying mechanistic framework that integrates the need for this departure from the prokaryotic enzymes. A discussion on this would enrich the manuscript. For example, could this have something to do with that the substrate alcohol in this case also has two long chain acyl groups and thus is constrained to be in the membrane, thereby imposing constraints on the relative positioning of the reactive groups ? 4. It seems that among the prokaryotic counterparts, the most similarity is with PgsA. In fact, the proposal in Fig. 5a Fig.12d. Typo in figure caption. Please omit xlCHPT1 from "…..and RsPIPS xlCHPT1".

Supplementary
3. The methodology section on cryoEM data processing could be improved by including the data processing parameters in various steps as well as adding them in the data processing scheme in supplementary figure section where appropriate.

POINT-BY-POINT RESPONSE TO REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this work the integral membrane Xenopus CHPT1 protein structure was determined by cryoEM to a resolution of 3.2-3.6 Angstroms. Human CHPT1 has been determined to synthesize the major membrane phospholipid phosphatidylcholine (PC) almost exclusively and is localized to the Golgi. Two other similar enzymes have been identified in humans, CEPT1 which can synthesize PC and phosphatidylethanolamine (PE) in the ER, and a PE specific EPT1.
Xenopus CHPT1-His6x was over-expressed and then purified from baculovirus using Co2+ resin, excision of the 6x-His tag, and then size exclusion chromatography prior to cryoEM. The structure was solved in the presence of the substrate CDP-choline as well as with CDP and the required cofactor Mg2+. The purified protein displayed a capacity to synthesize both PC and PE in an in vitro enzyme assay, with a strong preference for PC. The various CHPT1 variants produced were purified using a similar protocol and structure-activity relationships were probed by modelling prediction, site-directed mutagenesis, and enzyme activity assays. Substrate binding and catalytic residues were identified, including probing the CDP-aminoalcohol catalytic motif. This is the first experimentally resolved structure for a CHPT1 and many new molecular aspects by which the major membrane phospholipid PC (i.e. the synthesis of eukaryotic membranes) were described. Comments 1. I would suggest a paragraph/figure on how the cryoEM determined structure compares to that predicted by AlphaFold2 AlphaFold2 does a very good job predicting the structure, including features that are absent in the structures of the bacterial homologs, such as the parallel beta sheet on the intracellular side and the location of the amphipathic helix. There are only minor differences between the prediction and the structure. We added the model alignment in supplementary figure 7 and made a comment in Discussion.
2. On page 3 (Function of CHPT1) the first few sentences make it unclear if the CHPT1 from Xenopus is 68% identical and 83% similar to both CHPT1 and CEPT1. As these are different genes I would separate and discuss percent similarity to human CHPT1 and human CEPT1 in separate sentences to clarify.
Xenopus CHPT1 is 68% identical to both human CHPT1 and human CEPT1, and is 83% similar to both human CHPT1 and human CEPT1. We modified the sentence in question for clarity.
3. Methods (Cloning, expression and purification of Xenopus laevis CHPT1) -"condon" should be "codon" Fixed 4. Methods (Cloning, expression and purification of yeast pyrimidine…) -again, "condon" should be "codon" Fixed 5. Methods (Model building and refinement) -I believe "ploy-alanine" should be "poly-alanine" Fixed 6. I might suggest a title with more gravitas, perhaps along the lines of "Determining how phosphatidylcholine is synthesized by structure and mechanistic studies of a eukaryotic cholinephosphotransferase" We changed the title to "Structure of a eukaryotic cholinephosphotransferase-1 reveals mechanisms of substrate recognition and catalysis".
Reviewer #2 (Remarks to the Author): In this manuscript, the authors solve the first structure of a eukaryotic CHPT1, which carries out the final step of the Kennedy pathway of phosphatidylcholine biosynthesis. This is clearly an important target and is also a member of the CDP-alcohol superfamily of phosphatidyltransferases. The eukaryotic members have a different architecture than their prokaryotic counterparts and thus this work fills up an important void.

Key findings
The authors present two cryoEM structures of Xenopus laevis CHPT1, with CDP and CDP-choline bound in the active sites along with two Mg+2 ions. Although structures of related prokaryotic enzymes had been solved, the new structures reveal a novel architecture and enable the authors to propose a structure based mechanism of catalysis.